JPH0832637B2 - Synthetic immunogen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunogen comprising at least one antigen or antigenic determinant bound to a carrier, a compound containing an amphipathic adjuvant, the immunogen and the amphiphile. The present invention relates to a method for producing a compound containing a sex adjuvant and a pharmaceutical preparation (pharmaceutical composition) having immunizing activity and containing the immunogen.

In humans and animals, inoculation of non-self-derived antigens can generate an immune response. It is particularly utilized in protecting humans and animals from infection by pathogenic organisms such as viruses, bacteria, parasites, etc., but also in stimulating the production of antibodies to non-pathogenic substances, such as diagnostic tests and immunology. It is also used to produce antisera for use in sterilization or violence.

Such active immunization is usually carried out by inoculating the subject or animal to be protected with an inactivated or attenuated strain of the pathogen.

One of the disadvantages of inoculating inactivating agents is that the immunity is often reduced due to the partial denaturation of the antigen that occurs during inactivation. Inoculation with attenuated strains entails the risk that these strains still carry some virulence or are re-pathogenic due to spontaneous mutations.

For the above reasons, there is an increasing demand for better alternatives. As part of this, the use of immunogens, which consist in particular of antigens or antigenic determinants bound to carriers, has been carried out. In this way, antigens or even antigenic determinants, which themselves do not give rise to an immune response, can become active as immunogens.

The various carriers used for this purpose usually have drawbacks that make them difficult to use in general, eg in humans, such as toxicity and potential for cross-reactivity with endogenous substances.

An object of the present invention is to eliminate such drawbacks.

The immunogen of the present invention mainly contains at least one amphipathic adjuvant-associated molecule (eg, micelle), and the antigen and / or the antigenic determinant may be covalently bound to at least a part of these amphipathic adjuvants. Characterize.

The antigen and / or antigenic determinant of the immunogen of the present invention may be, for example, a polypeptide, protein, protein fragment, oligosaccharide or polysaccharide, oligonucleotide or polynucleotide, or other antigenic substance (compound).
These may be derived, for example, from pathogenic organisms such as viruses, bacteria, parasites, and in some cases may consist of fragments of antigens derived from these organisms, or synthesized to correspond to natural antigens or antigenic determinants. Alternatively, it may be derived from these naturally occurring antigens or determinants. The immunogen of the present invention may also include an antigen or antigenic determinant corresponding to, for example, a hormone or a hormone analog or a specific drug. Further, the antigen may be an anti-idiotype antibody or suitable fragment thereof. Such anti-idiotypic antibodies resemble an antigen or antigenic determinant with respect to relevant immunological properties.

Antigens and antigenic determinants can be isolated from biological or microbiological samples and then purified if desired. It can also be obtained by isolating larger molecules from the sample and cleaving or fragmenting them, where appropriate.

Furthermore, particularly low molecular weight antigens and antigenic determinants can also be obtained advantageously by chemical synthesis. This method can be used very easily in the case of small polypeptides, oligonucleotides and oligosaccharides, for example.

Such small antigen molecules, but particularly larger molecules, may also be advantageously produced using cell culture strains or microbial culture strains containing recombinant DNA encoding the production of the particular molecule. Also, using recombinant DNA technology, an antigen molecule containing multiple antigenic determinants present in various natural antigens, for example derived from multiple different pathogens or derived from multiple variants of one pathogen. Can also be manufactured.

In this case, the antigenic determinant means a chemical structure (body) capable of forming a recognition site for one or more kinds of antibodies directed thereto. Thus, the antigenic determinant in this case may consist of more than one epitope. Antigenic determinant also refers to so-called haptens, ie, independently existing chemical entities that, because of their relatively small size, cannot act as immunogens unless bound to a carrier.

Specific examples of suitable amphipathic adjuvants that can be used in the immunogens of the present invention include abridine (avridine, N, N
-Dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine), lipoidal amine (lipo
idal amine) 4-aminomethyl-1- (2,3- (di-n
- decyloxy)-n-propyl) -4-phenylpiperidine, dimethyl - dioctadecyl ammonium bromide, lauryl - muramyl - dipeptide, lauryl tetrapeptide (N ² - [N- (N- lauryl -L- alanyl)-gamma-D -Glutamyl] -N ^6- (Glycyl) -D, D
-L, L-2,6-diaminepimelamic acid, L-tyrosine and its alkyl derivative, maltos-tetrapalmitate,
Pluronic polyols, L-tyrosine-azobenzene-p-arsonate, sorbitan monooleate (Span 80), trehalose derivatives (trehalose dimycolate, etc.), retinoic acid and its derivatives, D, L-α- Tocopherols (vitamin E), lipid A and related glycosides such as saponins (Quillaja saponaria
Molina) from the bark) and Carbopol 934 ^(R) , Carbopol 940 ^(R) , Carbopol 941 ^{(R) and} other carbomers, as well as hydrophobic and hydrophilic Mention may be made of copolymers of phenyl derivatives.

The immunogens of the present invention include a molecular compound as described above, or a large number of said molecules associated such as, for example, micellar complexes, which may have different shapes and / or sizes.

The association molecule (eg, micelle) may be formed by an interaction between a plurality of homophilic amphipathic adjuvants or an interaction between two or more different amphipathic adjuvants.

In the immunogens of the invention, the antigen and / or antigenic determinant is covalently attached to at least a portion of the amphipathic adjuvant.

The nature of this binding depends on the amphipathic adjuvant and the groups involved in this binding on the antigen and / or antigenic determinant, as well as on the nature of the particular reaction component itself. This binding may occur directly between said group of antigens or antigenic determinants and said group of adjuvant molecules, or there may be a so-called "linker" between the antigens and antigenic determinants and adjuvant molecules. Can occur indirectly.

The immunogen of the present invention may be composed of an association molecule (eg, micelle) that binds to one antigen or antigenic determinant, or an association molecule (eg, micelle) that binds to at least two different antigens and / or antigenic determinants. . In the latter case, different species may represent the characteristics of one particular pathogen or substance against which it is desired to immunize (counterimmunity) or, if necessary, various variants of this substance, or a plurality of unrelated ones. Of different types may be characterized by pathogens and / or other substances against which immunity against them is desired.

The immunogens of the invention as described above can be stabilized if desired. Such stabilization may be achieved, for example, by binding at least a portion of the antigen and / or antigenic determinant and / or at least a portion of the amphipathic adjuvant to each other. In addition, the immunogen, whether bound or not, may be encapsulated and used for stabilization. Suitable capsules include, for example, those consisting of polymers that are degradable in the body. Such polymers include polylactic acid, polyglycolic acid, mixed polymers of lactic acid and glycolic acid, polyamino acids, mixed polymers of lactic acid and one or more amino acids, polymerized albumin and the like.

 Various methods can be used to produce the immunogens of the invention.

As an example, one may start with a compound (compound) of at least one antigen and / or antigenic determinant and at least one amphipathic adjuvant. The antigen and / or antigenic determinant and the amphipathic adjuvant may be linked together by one or more linkers if desired. Derivatives of such amphipathic adjuvants also form part of the invention.

The immunogen of the present invention is prepared by forming an associated molecule (for example, micelle) from a compound of this kind by a method usually used in the case of the compound (compound) as described above, and then separating the immunogen. Can be manufactured from To that end, it is possible to start from one compound containing at least one antigen and / or antigenic determinant and at least one amphiphile, but a compound mixture containing at least two different antigens and / or antigenic determinants. It is also possible to produce the immunogen from

Preferably, the micelles are prepared by sonicating a solution containing a derivative of an amphipathic adjuvant as described above at least in an amount equal to the critical micelle concentration. If desired, the adjuvant derivative can also be mixed with a corresponding or different free amphipathic adjuvant and from this mixture an immunogen comprising mixed micelles consisting partially of unbound adjuvant molecules can be prepared. .

In another method for producing the immunogen of the present invention, an antigen and / or an antigenic determinant is added by a method usually used in a covalent reaction,
The immunogen is isolated by covalent attachment to associated molecules (eg micelles) which consist primarily of amphipathic adjuvants.
This coupling is carried out through the reaction between the reactive groups of the adjuvant micelle and the reactive groups of the antigen and / or the antigenic determinant.

If desired, the amphipathic adjuvant, antigen and / or antigenic determinant can have a linker containing a reactive group.

Suitable reactive groups include amine groups, carboxyl groups,
Examples thereof include a hydroxyl group, a thiol group, a disulfide group, a carbonyl group, a maleimide group and an activated carboxyl group.

The immunogens of the invention may be stabilized (eg by the mutual binding of adjuvant molecules, antigens and / or antigenic determinants) and / or protected from digestion by hydrolysis (eg conjugated or not) regardless of the method of preparation. The immunogen may be encapsulated in a capsule, such as a polymer that can be degraded in the body.

The cross-linking of the antigen, the antigenic determinant and / or the amphipathic adjuvant can be generated using any reagent having at least two reactive groups suitable for reaction with the component to be bound. In the case of conjugation of polypeptides, for example, glutaraldehyde or formaldehyde or homobifunctional reagents (bisamidate or bisuccinimidyl ester) or heterobifunctional (he
It is advantageous to use a bifunctional reagent such as a terobifunctional) reagent (for example a compound having an azidophenyl group and a maleimide group, an amidate group, a succinimidyl group, a haloketone group or an activated fluorobenzene group).

Specific examples of polymeric materials that can be digested in the body and are suitable for encapsulation of immunogens have already been listed. These materials may be applied on or around the immunogen in conventional manner suitable for this purpose.

The immunogens of the invention are particularly suitable for use as a vaccine.

Such vaccines are mainly so-called "priming" (form the body to form specific free antibodies) when immunizing humans or animals against certain pathogens (viruses, bacteria, parasites etc.) or allergens. For pre-conditioning so that a strong immune response is elicited after a subsequent infection or re-vaccination, rather than directly stimulating for, or for example, immunological sterilization or castration (to the reproductive process). It can be used to carry out the required substances, eg raising antibodies against certain hormones.

Said vaccine may, if desired, comprise for example two or more different antigens and / or antigenic determinants bound to the same micelle, or of the invention such that each carries one antigen or antigenic determinant of a particular type. It may include a mixture of two or more immunogens. These various antigens and / or antigenic determinants may represent various pathogens or the like or variants of the same pathogen.

In order to be able to use the immunogen of the invention for vaccination purposes, it must be converted into a form suitable for the administration of a particular pharmaceutical. For intramuscular or subcutaneous administration, the immunogen should be mixed with a suitable liquid, such as saline.

Aqueous media are also most suitable for intranasal or intraocular administration, eg in the form of a spray. In the case of local administration such as oral administration, usually, an immunogen and, for example, a glycolytic enzyme present in the oral cavity, a proteolytic enzyme present in the stomach, or a dosage form that temporarily protects from a detergent is used. There is a need.

Such protection may include, for example, encapsulating one or more of the immunogens of the invention together in a suitable substance, or deferring the immunogen.
layed-release and / or controlled-rele
ase) in a dosage form or chemically modifying the site sensitive to proteolytic enzymes in polypeptide antigens and / or antigenic determinants such that proteolytic cleavage does not occur or is significantly suppressed. Can be implemented by

Example 1 Production of thiolated Quil A A. Method by reductive amination K. Dalsgaard (Archiv fuer die gesamte Virusforschun
g) “Saponin Adjuvants” 4
4,243-254 (1974)), DEAE-Sephacel (Sep
45 mg of Kir A pre-purified on 0.1 ml / l of pH 8.0
Dissolve in 0.8 ml sodium phosphate buffer. The clear solution is diluted with 1.5 ml of methanol and then 60 mg of 2- (2-pyridyldithio) -ethylamine.HCl are mixed with a solution of the above solution in 0.2 ml of phosphate buffer. 0.05 ml of a 1 mol / l solution of sodium cyanoborohydride in methanol is added and the reaction mixture is stirred at room temperature for 20 hours. Acetic acid 0.05
After adding ml, Sephadex in 0.1 mol / l sodium phosphate buffer, pH 6.0 containing 5 mMol / l EDTA
Low molecular weight components are removed by gel filtration using G-25.

Thereafter, dithioerythritol is added to the fraction containing Kill A until the concentration reaches 20 mmol / l. After standing at room temperature for 20 minutes, Sephadex G-25 in the above phosphate / EDTA buffer.
The thiolated Kill A is isolated by gel filtration through.

B. Method by amidation 10 mg of purified Kir A are dissolved in 0.2 ml of water and 0.2 ml of dimethylformamide. 1 mol / l HCl of this solution
The pH was brought to 3-3.5 and then 6.0 mg of 1-ethyl-3- (3
-Dimethylaminopropyl) -carbodiimide.HCl is added with stirring. After 3 minutes at room temperature 2- (2-pyridyldithio) -ethylamine.HCl 7.0 mg was added to pH 8.0 at 0.2 mol / l.
Add what is dissolved in 0.2 ml of sodium phosphate buffer. Add ethyldiisopropylamine and add pH to this solution.
To 8. After 30 minutes, add acetic acid until the pH is 5-6, then gel through Sephadex G-25 in 0.1 mol / l sodium phosphate buffer, pH 6.0 containing 5 mMol / l EDTA. Isolate derivatised Kil A.

Similar to the method A, reduction with dithioerythritol and separation of thiol-killed A are carried out.

C. Method by activation with 2,2,2-trifluoroethanesulphonyl chloride 50 mg of Kir A, purified as in A, are dissolved in dry pyridine and the solution is evaporated under vacuum. Repeat this operation again. The Kir A thus obtained was suspended in dry dioxane (0.5 ml) and then 8 μl of 2,2,2
-Add trifluoroethanesulfonyl chloride with vigorous stirring. After stirring for 10 minutes at room temperature, the mixture is diluted with 5 ml of 0.05 mol / l HCl precooled to 4 ° C. and then dialyzed against 0.01 mol / l HCl (2 × 1) at 4 ° C. for 5 hours.

To the resulting Kill A solution is added at 40C a solution of 40 mg of 2- (2-pyridyldithio) -ethylamine.HCl dissolved in 1 ml of 0.5 mol / l sodium phosphate buffer, pH 8.0. NaO
The pH was brought to 8.0 by addition of H and the mixture was then allowed to stand at 4 ° C.
Stir for 18 hours. 0.2 mol at pH 6.0 with 5 mmol / l EDTA
Derivatized Kil A is separated by gel filtration using Sephadex G-25 in / l phosphate buffer.

Similar to the case of A, reduction with dithioerythritol and separation of thiol-killed A are carried out.

Example 2 β-Endorphin- (6-17) -Immunogen I A. Preparation of activated abridine Trifluoromethanesulfonic anhydride 2.82 g (10 mmol)
Was dissolved in 20 ml of methylene chloride to give a solution of N, N-dioctadecyl-N'N'-bis- (2-hydroxyethyl).
-Propanediamine [Abridine: CP 20961] 1.67 g (25
(m mol) is added. Add 0.4 ml (5.2 mmol) of pyridine,
The mixture is stirred at room temperature for 60 minutes. A solution of 2.5 g (32 mmol) of thiolacetic acid in water and pH adjusted to 7.5 with NaOH is added. The two-phase system is vigorously stirred for 60 minutes. The methylene chloride layer was separated, dried over MgSO _4, and evaporated.
The residue is recrystallized from methylene chloride / ether.

The abridine S-acetyl derivative in methylene chloride is mixed with abridine (1: 1). The solvent was evaporated and the residue was adjusted to pH 7.0 with 0.1 mol / l NaCl and 0.05 mol / l EDTA.
Disperse in 0.1 mol / l sodium phosphate buffer (5 mg
/ ml). Sonicate this dispersion under N _{2 at} 60 ° C. for 5 minutes [Branson Sonifier;
Microtip, 20W].

B. Preparation of β-Endorphin- (6-17) -Immunogen 0.5 mol / l hydroxylamine, pH 7.2, deoxygenated to the solution of derivatized abridine prepared in Example A to a final concentration of 0.04. Add until mol / l. The mixture is maintained at 35 ° C. for 30 minutes under N ₂ atmosphere. afterwards,
1.5 equivalents of β-endorphin- (6-17) maleimide derivative to thiolated abridine in 0.1 mol / l sodium phosphate buffer of pH 6.0 containing 0.005 mol / l EDTA The solution in which was dissolved is added at 20 ° C.

The mixture was stirred under N ₂ for 15 hours, then 0.1 mol / l
Dialyze against 0.05 mol / l sodium phosphate buffer, pH 7.4, containing NaCl.

Example 3 β-Endorphin- (6-17) -Immunogen II 1.2 molar equivalents (based on thiol groups) of β- in 0.1 mol / l phosphate buffer pH 6.0 containing 5 mmol / l EDTA. Endorphin- (6-17) maleimide derivative, ie Is added to the solution of thiolated Kill A prepared in 1A (7 mg / ml).

The mixture is allowed to react at room temperature for 1 hour, then 150 mmol
Dialyze for 15 hours at 0 ° C. against 0.02 mol / l sodium phosphate buffer, pH 7.4 containing / ml NaCl.

Example 4 Parvovirus Immunogen A. Production of Porcine Parvovirus Antigen A 65 kD coat protein of porcine parvovirus (PPV) was added to Moritor, T-Double (Molitor, TW), Joo, Et S (Joo, HS) and J. Virology 4 by Collet, MS
Isolate according to the method disclosed in 5,842-854 (1983). -Concentrated CaCl ₂ solution was added to the tissue culture supernatant containing PPV to a CaCl ₂ concentration of 25 mmol / l, -The resulting virus precipitate was centrifuged at 12,000 xg for 20 minutes, -Pellet containing virus Is subjected to CsCl gradient centrifugation to separate a fraction having a density of 1.3 g / ml, dialyzed and treated with 1% sodium dodecyl sulfate (SDS) for 3 minutes, and a 65 kD protein is isolated by SDS / PAGE fractionation, It was dialyzed and lyophilized.

B. Derivatization of porcine parvovirus antigen The protein obtained in A is adjusted to pH 7.5 until the concentration reaches 3 mg / ml.
In 0.1 mol / l sodium phosphate buffer, then treated with 5 molar equivalents of N-succinimidyl-4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC) for 30 minutes at room temperature.

Excess SMCC is removed by gel filtration through Sephadex G-25 in 0.1 mol / l sodium phosphate buffer pH 6.0 containing 0.9% NaCl and 5 mmol / l EDTA.

C. Preparation of immunogen The derivatized PPV coat protein prepared in B was used in Example 1A.
1: 4 with thiolated Kill A obtained in (Kill A: protein)
Mix at a ratio of (weight / weight).

Keep this solution at room temperature for 5 hours, then 250,000 ×
Isolate the immunogen by centrifugation at 10-40% (w / w) sucrose gradient for 10 hours at + 4 ° C.

Then 0.02 mol / l at pH 7.4 containing 150 mmol / l NaCl
Dialyzing the PPV immunogen against phosphate buffer.

Example 5 Parvovirus Peptide Immunogen A maleimide derivative of a peptide corresponding to a partial sequence of the PPV coat protein (obtained from Amgen AMGen). Is attached to the thiolated Kill A by the method of Example 3.

Example 6 HBsAg Immunogen A polypeptide derived from HBsAg (hepatitis B surface antigen) lacking the amino-terminal hydrophobic moiety was labeled with Fujisaw.
a) According to another method (Nucleic Acids Res
earch) 11 , 3581-3590 (1983)) Manufactured using E. coli. The polypeptides extracted from these bacteria are purified by affinity chromatography.

Prior to conjugation to an adjuvant, SMCC is used to activate the purified HBsAg derivative as described in Example 4B.

Then, the activated polypeptide is bound to thiol-killed A by the same method as in Example 4C.

Example 7 Parvovirus Immunogen A. Preparation of Thiolated Kill A Kill A (20 mg) is dissolved in dimethylformamide (0.5 ml). HCl (10 μmol; 1N solution in DMF) is added and after cooling the solution to 0 ° C., carbonyldiimidazole (20 μmol in 0.04 ml DMF) is added. The reaction mixture is stirred at 0 ° C. for 30 minutes. 2- (2-Pyridyldithio) -ethylamine.HCl (50 μmol) and ethyldiisopropylamine (50 μmol) are added. The resulting clear solution is 18
Hold at room temperature for hours. DMF is removed by vacuum rotary evaporation and the resulting syrup is dissolved in water (2 ml). 5 mmol /
Derivatized Kil A is separated by gel filtration on Sephadex G-25 in 0.1 mol / l sodium phosphate pH 6.0 also containing 1 EDTA. Reduction with dithioerythritol and gel filtration with Sephadex G-25 as described in Example 1A gives the thiolated Kill A.

B. Preparation of Derivatized Porcine Parvovirus Antigen A solution of parvovirus protein (2 mg) isolated by the method of Example 4A in 0.1 mol / l sodium phosphate, pH 7.6 was dissolved in N-succinimidyl 3- (2 -Pyridyldithio) -propionate (SPDP; in 0.3 ml ethanol 3.
2 μmol) at room temperature for 3 hours. 5 mmol / l EDT
The 2-pyridyl disulfide substituted protein is separated by gel filtration on Sephadex G-25 in 0.1 mol / l sodium phosphate, pH 6.0, which also contains A.

C. Preparation of Immunogen A solution of thiolated Kill A (containing 0.6 μmol of free thiol group) obtained by the method of A was added to a 2-pyridyldisulfide-substituted porcine parvovirus protein (0.45 μm) obtained by the method of B. Mol of 2-pyridyl disulfide groups).

The binding reaction is monitored by measuring the release of pyridine-2-thione and is complete within 2 hours. The Kill A-protein conjugate is dialyzed against 0.02 mol / l sodium phosphate, pH 7.4, which also contains 150 mmol / l NaCl.

Example 8 Immunogen efficacy Two groups of 10 Swiss albino mice 6 weeks old were repeatedly inoculated ip with a rabies virus coat peptide linked to either KLH or Kill A.

This peptide has the amino acid sequence Asp-Tyr-Arg-Trp-Leu-Arg-Thr-Val-Lys-Thr-Thr-Lys-Gl.
It has y-Ser. Each mouse was repeatedly inoculated with 50 μg of the peptide bound to either 50 μg of Kill A or 500 μg of KLH.

The sera of these mice were diluted 1/80 and tested by the enzyme-labeled immunosorbent assay (ELISA) using the following test system after the third and fourth inoculations, respectively.

After the third inoculation: virus-coated microtiter plate-mouse serum-anti-mouse Ig-HRP-
Zygote.

After the 4th inoculation: microtiter plate coated with human anti-rabies monoclonal antibody-ERA virus antigen-mouse serum-anti-mouse Ig-HRP-conjugate.

 The results are shown in the table below.

Serum response of mice inoculated with the following peptides (450 nm)
After the 3rd inoculation After the 4th inoculation KLH-peptide 0.185 0.939 Quil-A peptide 0.200 0.997 control serum 0.072 0.240 As is apparent from these results, the antibody generated against the kill A-peptide complex is an antibody against the KLH-peptide complex. At least as good as virus binding.

Example 9 Adjuvant Effect of Saponin Derivatives Pseudorabies (PR) antigens alone or with an adjuvant to be tested were im inoculated into 4 groups of 10 groups of 9 Swiss albino mice 9 weeks old.

Serum was collected after 3, 9 and 15 weeks and pooled to test anti-PR response by enzyme linked immunosorbent assay (ELISA).

 The results are shown in the table below.

These results show that the adjuvant activity of Kil A persists after derivatization of the adjuvant and even after conjugation of the peptide.

Claims (11)

[Claims] 1. A carrier comprising at least one antigen and / or an antigenic determinant bound to a carrier, the carrier mainly comprising at least one amphipathic adjuvant molecule associated molecule, at least a portion of said adjuvant molecule. Is covalently linked to the antigen and / or antigenic determinant. 2. At least one antigen and / or antigenic determinant
Immunogen according to claim 1, characterized in that it is linked to the amphipathic adjuvant molecule via one linker. 3. The immunogen according to claim 1, which comprises saponin as an amphipathic adjuvant. 4. A plurality of different types of antigens and / or antigenic determinants are contained, and these antigens and / or antigenic determinants are applied to a plurality of different types of pathogens and / or other substances for which counterimmunity is desired. The immunogen according to any one of claims 1 to 3, which is unique. 5. The method according to any one of claims 1 to 4, wherein at least a part of the antigenic determinant and / or the antigen and / or the amphipathic adjuvant molecule is bound to each other. Immunogen. 6. The immunogen according to any one of claims 1 to 5, which is encapsulated in a capsule. 7. An immunogen consisting of at least one antigen and / or an antigenic determinant bound to a carrier, the carrier consisting essentially of an associated molecule of at least one amphipathic adjuvant molecule, said adjuvant molecule comprising: A method of making said immunogen, at least a portion of which is covalently bound to said antigen and / or antigenic determinant, wherein at least one free amphipathic adjuvant is present, if desired, and at least one amphipathic Forming an associated molecule from a compound containing at least one kind of amphipathic adjuvant molecule, which comprises a glycoside and at least one antigen and / or an antigenic determinant, and which are covalently bonded to each other, by a conventional method suitable for the purpose; The immunogen thus formed is then isolated, after which it may be stabilized and / or hydrolyzed if desired. Wherein the protecting against sexual cleavage. 8. A compound containing an amphipathic adjuvant molecule,
Claims, characterized in that they are obtained by combining at least one amphipathic adjuvant molecule with at least one antigen and / or antigenic determinant by methods commonly used for compounds of this type. The method according to item 7. 9. An immunogen consisting of at least one antigen and / or an antigenic determinant bound to a carrier, which carrier mainly consists of an associated molecule of at least one amphipathic adjuvant molecule, A method for producing the immunogen, at least a part of which is covalently bound to the antigen and / or antigenic determinant, wherein the antigen and / or antigenic determinant optionally has at least one linker, and optionally one or more. With an associative molecule mainly comprising at least one amphipathic adjuvant, and reacting with the above-mentioned antigen and / or antigenic determinant or a linker which may be bound to these, , Forming a covalent bond with the amphipathic adjuvant or a linker which may be bound thereto, and then The formed an immunogen is isolated, wherein the protecting the immunogen against stabilized and / or hydrolytic cleavage, if subsequently desired. 10. An immunogen consisting of at least one antigen and / or an antigenic determinant bound to a carrier, the carrier consisting mainly of an associated molecule of at least one amphipathic adjuvant molecule, said adjuvant molecule comprising: An immunologically active pharmaceutical composition comprising a plurality of said immunogens, at least a portion of which is covalently bound to said antigens and / or antigenic determinants, in a delayed release dosage form. 11. An immunogen comprising at least one antigen and / or an antigenic determinant bound to a carrier, the carrier comprising predominantly at least one associative molecule of an amphipathic adjuvant molecule, said adjuvant molecule comprising: A mixed vaccine formulation, characterized in that it comprises two or more different types of said immunogens, at least a portion of which is covalently linked to said antigens and / or antigenic determinants.